Bioprocess Engineering Aspects of Biopolymer Production by the Cyanobacterium <i>Spirulina</i> Strain LEB 18

Martins, Roberta Guimarães; Severo Gonçalves, Igor; de Morais, Michele Greque; Costa, Jorge Alberto Vieira

doi:https://doi.org/10.1155/2014/895237

International Journal of Polymer Science

On this page

Abstract Introduction Methods Results and Discussion Conclusions References Copyright Related Articles

Research Article | Open Access

Volume 2014 | Article ID 895237 | https://doi.org/10.1155/2014/895237

Bioprocess Engineering Aspects of Biopolymer Production by the Cyanobacterium Spirulina Strain LEB 18

Roberta Guimarães Martins,¹Igor Severo Gonçalves,¹Michele Greque de Morais,¹and Jorge Alberto Vieira Costa¹

Academic Editor: Saad Khan

Received19 Aug 2014

Revised15 Nov 2014

Accepted24 Nov 2014

Published11 Dec 2014

Abstract

Microbial biopolymers can replace environmentally damaging plastics derived from petrochemicals. We investigated biopolymer synthesis by the cyanobacterium Spirulina strain LEB 18. Autotrophic culture used unmodified Zarrouk medium or modified Zarrouk medium in which the NaNO₃ content was reduced to 0.25 g L⁻¹ and the NaHCO₃ content reduced to 8.4 g L⁻¹ or increased to 25.2 g L⁻¹. Heterotrophic culture used modified Zarrouk medium containing 0.25 g L⁻¹ NaNO₃ with the NaHCO₃ replaced by 0.2 g L⁻¹, 0.4 g L⁻¹, or 0.6 g L⁻¹ of glucose (C₆H₁₂O₆) or sodium acetate (CH₃COONa). Mixotrophic culture used modified Zarrouk medium containing 0.25 g L⁻¹ NaNO₃ plus 16.8 g L⁻¹ NaHCO₃ with the addition of 0.2 g L⁻¹, 0.4 g L⁻¹, or 0.6 g L⁻¹ of glucose or sodium acetate. The highest biopolymer yield was 44% when LEB 18 was growing autotrophically in media containing 0.25 g L⁻¹ NaNO₃ and 8.4 g L⁻¹ NaHCO₃.

1. Introduction

Instability in the international price of oil and natural gas, coupled with geopolitical factors and environmental concerns, has led to the need to substitute nonrenewable petroleum products with those based on renewable resources. The production of polymers from petrochemicals is the second major use of oil after their utilization as a source of energy [1]. Biopolymers can be produced using biofixation of carbon dioxide by cyanobacterium and could reduce both dependency on petroleum and carbon dioxide emissions [2].

Petrochemical-based plastics are resistant to degradation and most eventually end up in sanitary landfills, where they often compromise the circulation of gas and liquids and thus the decomposition of other materials within the site and may even make it unstable. Since landfill sites are becoming scarce, one solution is to substitute recalcitrant petrochemical-based plastics with biopolymers that do not cause such problems [3].

Polyhydroxyalkanoates (PHAs), including polyhydroxybutyrate (PHB), polyhydroxypropionate (PHP), and polyhydroxyvalerate (PHV), are bacterial or cyanophyte aliphatic polyesters which have thermoplastic, mechanical, and physical properties similar to polypropylene. PHAs are biocompatible, recyclable, and biodegradable and produce zero toxic waste since they biodegrade into carbon dioxide and water by microbial attack in about three months to one year [4, 5]. These polymers have a high degree of polymerization and are crystalline, optically active, isotactic, and insoluble in water [6, 7].

Cyanobacteria have the potential for the production of biopolymers and their yield can be increased by stressing the culture via nutrient limitation or other means, use of recombinant strains [8], control of metabolic flux, and the use of different bioreactor types [1]. Some cyanophytes can adapt their metabolism during nutrient limitation [9], with biopolymer synthesis generally occurring when the carbon and energy source is present at normal levels or in excess while at least one other nutrient is limiting, nitrogen, phosphorus, magnesium, and iron being among the most common limiting nutrients [3]. Unlike crop plants, the cultivation of cyanobacteria does not require the use of large areas of ground and can occupy areas inappropriate for agriculture and thus does not compete with food production [10].

We investigated methods to stimulate the synthesis of biopolymer by the cyanophyte Spirulina strain LEB 18 cultivated with different carbon sources and reduced nitrogen levels.

2. Material and Methods

2.1. Microorganism and Culture Media

The cyanobacterium Spirulina strain LEB 18 [11] was maintained in unmodified Zarrouk liquid mineral salts medium, containing mineral salts (K₂HPO₄, K₂SO₄, NaCl, MgSO₄, CaCl₂, FeSO₄, and EDTA) and 16.8 g L⁻¹ of sodium bicarbonate (NaHCO₃) as the carbon source and 2.5 g L⁻¹ of sodium nitrate (NaNO₃) as the nitrogen source [12].

Autotrophic culture used unmodified Zarrouk medium or modified Zarrouk medium in which the NaNO₃ content was reduced to 0.25 g L⁻¹ and the NaHCO₃ content was reduced to 8.4 g L⁻¹ or increased to 25.2 g L⁻¹. Heterotrophic culture used modified Zarrouk medium containing 0.25 g L⁻¹ NaNO₃ with the NaHCO₃ replaced by 0.2 g L⁻¹, 0.4 g L⁻¹, or 0.6 g L⁻¹ of glucose (C₆H₁₂O₆) or sodium acetate (CH₃COONa). Mixotrophic culture used modified Zarrouk medium containing 0.25 g L⁻¹ NaNO₃ plus 16.8 g L⁻¹ NaHCO₃ with the addition of 0.2 g L⁻¹, 0.4 g L⁻¹, or 0.6 g L⁻¹ of glucose or sodium acetate. The growth media components are summarized in Table 1.

2.2. Experimental Conditions

Cultures were grown in 2 L closed photobioreactors with a working volume of 1.5 L and continuous agitation provided by the injection of sterile air. The inocula were adapted for 20 days in their respective culture media. The initial biomass concentration was 0.15 g L⁻¹ and the initial volume was 1.5 L. The cultures were maintained at 30°C for 15 days under a 12 h photoperiod and 3200 lux provided by 40 W daylight-type fluorescent lamps.

2.3. Analytical Assays

Samples (10 mL) were collected aseptically at the same time every 24 h and the pH was measured at the same time using a Q400H digital pH meter (Quimis, Brazil) according to the methodology of the Association of Analytical Communities [13]. Growth of LEB 18 was estimated by measuring the optical density at 670 nm in a Q798DRM spectrophotometer (Quimis, Brazil) and comparing the reading to a calibration curve relating optical density to biomass [14].

2.4. Determination of Kinetic Parameters

Growth curves of LEB 18 biomass against time were plotted and the following parameters calculated: maximum biomass concentration (, g L⁻¹); productivity (, g L⁻¹ d⁻¹), calculated as [15], in which is the biomass (g L⁻¹) at time (d) and is the biomass (g L⁻¹) at time (d), maximum productivity (, g L⁻¹ d⁻¹) being the productivity at , and specific growth rate (, d⁻¹) was calculated as , where 0.693 is the natural logarithmic of 2 and is the biomass doubling time [16], the maximum specific growth rate being (d⁻¹).

2.5. Extraction Biopolymers

After fifteen days of cultivation, biopolymers were extracted from each photobioreactor run by centrifuging the culture media to precipitate the LEB 18 biomass, to which was added 10% to 12% v/v sodium hypochloride solution and the mixture recentrifuged. The supernatant was discarded and the precipitate was washed with distilled water, then recentrifuging and again discarding the supernatant and adding acetone to precipitate the biopolymer. Then, it was dried in an oven at 35°C for 48 h.

The yield () was calculated as grams of biopolymer (bp) per gram of biomass (bm) from the equation , where is the final amount of biopolymers in grams extracted from LEB 18 and is the amount of LEB 18 biomass in grams from which the biopolymers were extracted.

2.6. Statistics Analysis

Where appropriate, the data was subjected to analysis of variance (ANOVA) and the Tukey test at %. All reagents were of at least analytical grade. Where appropriate, percentages are weight dry for weight dry (w/w) unless otherwise indicated.

3. Results and Discussion

The LEB 18 data is given in Table 1 and the growth curves are given in Figures 1 and 2, from which it can be seen that the different culture media were broadly similar regarding the various carbon sources (Table 1). The curves show no lag phase (Figures 1 and 2) because the inocula were preadapted for each culture medium, thus allowing LEB 18 to enter directly into the exponential growth phase. Growth, measured as , was not limited during the exponential phase, even in the experiments containing the maximum concentration of carbon (16.8 g L⁻¹ of glucose or sodium acetate, 25.2 g L⁻¹ of NaHCO₃).

(a)

(b)

(c)

(d)

The culture which obtained the highest value (0.86 g L⁻¹) used NaHCO₃ (8.4 g L⁻¹ and 16.8 g L⁻¹) as the carbon source and 0.25 g L⁻¹ NaNO₃ as the nitrogen source (Table 1, Figure 2). The highest value (0.12 d⁻¹) occurred with unmodified Zarrouk medium (Table 1). However, there was no statistical difference between any of the culture media regarding values (Table 1). Since bicarbonate is the carbon source in unmodified Zarrouk medium, LEB 18 already expressed the enzymes necessary for carbon assimilation from this carbon source, enabling more growth than glucose or acetate.

The experiments were concluded after 15 days when the cultures entered the stationary phase. In general, the production of intracellular biocompounds generally occurs in the stationary phase when microbial growth ceases. However, biopolymers such as PHAs serve as intercellular energy reserves, because of which their production generally occurs during the exponential phase in parallel with whatever factor is used to measure cell growth, which in our case was the increase in LEB biomass. This aids in the survival of the producing microorganism, which can use PHAs for survival in the later stages of growth when nutrients become limiting.

We carried out preliminary tests in which we extracted biopolymers every 5 days for 30 days (data not shown) and observed that the end of the exponential phase and the beginning of the stationary phase occurred at around 15 days after inoculation and that biopolymer yields were highest at this time. Studies by other workers of biopolymer production by cyanophytes have shown higher yields during the stationary phase, with the cyanophyte Nostoc muscorum producing 8.6% [17] and the cyanophyte Synechocystis sp. strain PCC 6803 producing 45% during this phase.

The quantity of nitrogen available is known to directly influence biopolymer synthesis and in our experiments the biopolymer yield increased fourfold from 10.26% to 40% when the sodium nitrate content was reduced from 2.5 g L⁻¹ to 0.25 g L⁻¹, 10% of its original value (Table 1).

Unmodified Zarrouk medium contained 16.8 g L⁻¹ NaHCO₃ plus 2.5 g L⁻¹ NaNO₃ and produced a yield () of 10.26%, probably due to the high level of nitrogen (Table 1). However, in modified Zarrouk media containing the same quantity of NaHCO₃ but only 0.25 g L⁻¹ NaNO₃ the biopolymer yield was 40%. Furthermore, when growing in modified Zarrouk medium with reduced nitrogen, d⁻¹ was half the rate occurring with unmodified Zarrouk medium, where d⁻¹ (Table 1). It is known that values are higher for actively growing cells, which was the case in unmodified Zarrouk medium, and that when energy is diverted for the synthesis of storage compounds, including biopolymers, lower values of occur. It is interesting to note that while there was no statistically significant difference () between the values for the three versions of modified Zarrouk medium, there was a statistically significant difference () between these and the unmodified Zarrouk medium (Table 1).

It has been reported that the eukaryotic algae Nannochloropsis growing in wastewater supplemented with /2 medium showed d⁻¹ and a lipids content of 30% but when d⁻¹ the lipid content was only 23% [18]. Other workers have reported d⁻¹ during semicontinuous cultivation of the cyanophyte Cyanobium in BG-11 medium with the addition of 0.4 g L⁻¹ of NaHCO₃ [19].

The maximum productivity (, Table 1) occurred between the second and the eighth days of culture, probably because at this stage nutrients were available in greater concentrations and LEB 18 had been preadapted to the culture media (Figure 1(a)). Furthermore, during this initial period, little PHB would have been produced and the cultures energy resources could have been directed mainly to biomass production. In the trials with inorganic carbon, occurred between the fifth and the eighth days of culture. Our values ranged from 0.04 g L⁻¹ d⁻¹ to 0.17 g L⁻¹ d⁻¹ (Table 1), similar to the g L⁻¹ d⁻¹ reported for Cyanobium growing in BG-11 media [19] and g L⁻¹ d⁻¹ for LEB 18 growing in medium containing 10 g L⁻¹ NaHCO₃ [2].

The medium which presented the highest biopolymer yield () contained 8.4 g L⁻¹ NaHCO₃ and 0.25 g L⁻¹ of nitrogen, and there appeared to be a reduction in yield as the NaHCO₃ concentration increased in the reduced nitrogen media, although the differences were not statistically significant (Table 1). However, it is known that although high levels of carbon are needed to stimulate the synthesis of biopolymers the excess can also inhibit production because although acetyl-CoA can enter the PHB biosynthesis pathway free acetyl-CoA inhibits the enzyme -ketothiolase which is responsible for PHB synthesis.

The addition of the organic carbon sources, glucose, and sodium acetate did not stimulate either biomass production or biopolymer synthesis. Furthermore, there was no significant difference () in biopolymer yield when adding sodium bicarbonate to cultures containing glucose or sodium acetate. It is known that when two endogenous carbon sources are available a microorganism usually gives preference to one of them. Since LEB 8 is generally maintained in unmodified Zarrouk medium containing sodium bicarbonate it already has the necessary pathways to utilize this nutrient, making two sources of carbon superfluous. It has been reported that Spirulina platensis UMACC 161 can produce 10% PHB when using a medium containing 9 g L⁻¹ of NaHCO₃ plus 0.5% sodium acetate without the presence of nitrogen but when the media contained NaHCO₃ plus CO₂ the PHB yield was around 3% [20]. Yields of 47% PHB have been reported for the cyanophyte Nostoc muscorum growing in BG-11 medium containing glucose and sodium acetate but without a nitrogen source [21].

The biopolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) has been produced from Nostoc muscorum using various carbon sources (acetate, fructose, glucose, propionate, and valerate) with the PHBV yield being 28% when using 0.4% w/v acetate and 26% when using 0.4% w/v glucose, the yield rising to 60% when the carbon source was 0.4% acetate or valerate and the nitrogen source was restricted [22]. Lower yields of 9.5% have been reported for the cyanophyte Synechocystis sp. strain PCC 6803 growing in BG-11 medium. However, when cultivated with sodium acetate, the yield increased to 14.6% under nitrogen limitation and 25.7% under phosphate limitation [23].

The yield of microbial biopolymers is quite variable and depends on the composition of the medium used. When we grew LEB 18 on sodium bicarbonate, glucose, or sodium acetate along with different concentrations of nitrogen we obtained biopolymer yields varying from 7.64% to 44.19% (Table 1). The cyanophyte Aulosira fertilissima has been investigated for PHB production using media containing 0.2% w/v to 0.4% w/v acetate or citrate plus from zero to 20 mg L⁻¹ K₂HPO₄ with an incubation period from 2 days to 14 days, with PHB yields ranging from 17% to 85.5% [24].

Bacteria produce large amounts of biopolymers in a short time; however, cyanobacteria have the advantage of using smaller amounts of nutrients due to photosynthesis, which uses the solar energy and transforms the carbon dioxide into oxygen, which is essential for humans.

Decreasing the quantity of nitrogen in the culture medium resulted in an increase in polymer production by LEB 18. Autotrophic growth in modified Zarrouk medium containing 16.8 g L⁻¹ of NaHCO₃ and 0.25 g L⁻¹ of NaNO₃ resulted in about 74% more biopolymer production than in unmodified Zarrouk medium (Table 1). It has been pointed out that cyanophyte Spirulina is a rich source of proteins [25], implying a large nitrogen requirement for growth. Under nitrogen limitation it is known that cyanophytes can divert carbon into other metabolic routes and produce biopolymers [21] to serve as carbon and energy storage compounds which can be reused when conditions become more favorable. The nitrogen content of the environment increases and the organism can produce proteins for cell growth rather than the storage lipids from which PHB derives.

4. Conclusions

In our experiments we found that the maximum biopolymer yield was 44.19% when LEB 18 was cultivated in modified Zarrouk medium containing 8.4 g L⁻¹ of sodium bicarbonate and 0.25 g L⁻¹ of sodium nitrate. The biopolymers produced by cyanobacteria have many uses because they present biocompatibility with mammalian cells and tissues and biodegradability. The main applications for biopolymers are in the food and medical areas, but they can also help reduce the environmental pollution generated by petrochemical-derived plastics.

This study shows that the cyanobacteria are potential sources of biopolymers, because they can use smaller amounts of nutrients and help to reduce the environmental pollution.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

References

L. F. Silva, J. G. C. Gómez, R. C. S. Rocha, M. K. Tacino, and J. G. C. Pradella, “Produção biotecnológica de poli-hidroxialcanoatos para a geração de polímeros biodegradáveis no Brasil,” Química Nova, vol. 30, no. 7, pp. 1732–1743, 2007.
View at: Google Scholar
M. R. Andrade, F. V. Camerini, and J. A. V. Costa, “Perda química de carbono e cinética do crescimento celular em cultivos de Spirulina,” Química Nova, vol. 31, no. 8, pp. 2031–2034, 2008.
View at: Google Scholar
U. A. Lima, E. Aquarone, W. Borzani, and W. Schmidell, Biotecnologia Industrial—Processos Fermentativos e Enzimáticos, vol. 3, Edgard Blücher, São Paulo, Brazil, 2001.
V. A. Konagesli Jr., L. Dagostini, and M. Renz, Plásticos Biodegradáveis: Revisão Sobre Métodos de Preparação e Propriedades da Biodegradação, Departamento de Biologia e Química, Universidade Regional do Noroeste do Estado do Rio Grande do Sul, 2012, http://www.scribd.com/doc/3033642/PLASTICOS-BIODEGRADAVEIS.
H. Abe and Y. Doi, “Structural effects on enzymatic degradabilities for poly[(R)-3-hydroxybutyric acid] and its copolymers,” International Journal of Biological Macromolecules, vol. 25, no. 1–3, pp. 185–192, 1999.
View at: Publisher Site | Google Scholar
D. B. Hazer, E. Kiliçay, and B. Hazer, “Poly(3-hydroxyalkanoate)s: diversification and biomedical applications: a state of the art review,” Materials Science and Engineering C, vol. 32, no. 4, pp. 637–647, 2012.
View at: Publisher Site | Google Scholar
C. S. K. Reddy, R. Ghai, and V. C. Kalia, “Polyhydroxyalkanoates: an overview,” Bioresource Technology, vol. 87, no. 2, pp. 137–146, 2003.
View at: Publisher Site | Google Scholar
A. Shrivastav and S. K. Mishra, “Polyhydroxyalkanoate (PHA) synthesis by Spirulina subsalsa from Gujarat coast of India,” International Journal of Biological Macromolecules, vol. 46, no. 2, pp. 255–260, 2010.
View at: Publisher Site | Google Scholar
T. M. Mata, A. A. Martins, and N. S. Caetano, “Microalgae for biodiesel production and other applications: a review,” Renewable and Sustainable Energy Reviews, vol. 14, no. 1, pp. 217–232, 2010.
View at: Publisher Site | Google Scholar
S. Nonhebel, “Renewable energy and food supply: will there be enough land?” Renewable and Sustainable Energy Reviews, vol. 9, no. 2, pp. 191–201, 2005.
View at: Publisher Site | Google Scholar
M. G. Morais, C. D. Reichert, F. Dalcanton, A. J. Durante, L. F. Marins, and J. A. V. Costa, “Isolation and characterization of a new Arthrosphira strain,” Zeitschrift für Naturforschung C, vol. 63, no. 1-2, pp. 144–150, 2008.
View at: Google Scholar
C. Zarrouk, Contribution à l'étude d'une Cyanophycée, influence de diveurs facteurs physiques et chimiques sur la croissance et photosynthese de Spirulina maxima Geitler [Ph.D. thesis], University of Paris, Paris, France, 1966.
AOAC, Official Methods of Analysis, Method 923.05, chapter 32, Supplement, 15, Association of Analytical Communities, 1995.
J. A. V. Costa, M. G. De Morais, F. Dalcanton, C. Da Cruz Reichert, and A. J. Durante, “Simultaneous cultivation of Spirulina platensis and the toxigenic cyanobacteria Microcystis aeruginosa,” Zeitschrift fur Naturforschung C: Journal of Biosciences, vol. 61, no. 1-2, pp. 105–110, 2006.
View at: Google Scholar
J. E. Bailey and D. F. Ollis, Biochemical Engineering Fundamentals, McGraw-Hill, Singapore, 2nd edition, 1986.
W. Schmidell, A. U. Lima, E. Aquarone, and W. Borzani, Biotecnologia Industrial, vol. 2, Edgard Blücher, São Paulo, Brazil, 2001.
L. Sharma and N. Mallick, “Accumulation of poly-β-hydroxybutyrate in Nostoc muscorum: regulation by pH, light-dark cycles, N and P status and carbon sources,” Bioresource Technology, vol. 96, no. 11, pp. 1304–1310, 2005.
View at: Publisher Site | Google Scholar
L. Jiang, S. Luo, X. Fan, Z. Yang, and R. Guo, “Biomass and lipid production of marine microalgae using municipal wastewater and high concentration of CO₂,” Applied Energy, vol. 88, no. 10, pp. 3336–3341, 2011.
View at: Publisher Site | Google Scholar
A. A. Henrard, M. G. de Morais, and J. A. V. Costa, “Vertical tubular photobioreactor for semicontinuous culture of Cyanobium sp,” Bioresource Technology, vol. 102, no. 7, pp. 4897–4900, 2011.
View at: Publisher Site | Google Scholar
M.-H. Jau, S.-P. Yew, P. S. Y. Toh et al., “Biosynthesis and mobilization of poly(3-hydroxybutyrate) [P(3HB)] by Spirulina platensis,” International Journal of Biological Macromolecules, vol. 36, no. 3, pp. 144–151, 2005.
View at: Publisher Site | Google Scholar
L. Sharma, A. Kumar Singh, B. Panda, and N. Mallick, “Process optimization for poly-β-hydroxybutyrate production in a nitrogen fixing cyanobacterium, Nostoc muscorum using response surface methodology,” Bioresource Technology, vol. 98, no. 5, pp. 987–993, 2007.
View at: Publisher Site | Google Scholar
R. Bhati and N. Mallick, “Production and characterization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) co-polymer by a N₂-fixing cyanobacterium, Nostoc muscorum Agardh,” Journal of Chemical Technology and Biotechnology, vol. 87, no. 4, pp. 505–512, 2012.
View at: Publisher Site | Google Scholar
B. Panda, P. Jain, L. Sharma, and N. Mallick, “Optimization of cultural and nutritional conditions for accumulation of poly-β-hydroxybutyrate in Synechocystis sp. PCC 6803,” Bioresource Technology, vol. 97, no. 11, pp. 1296–1301, 2006.
View at: Publisher Site | Google Scholar
S. Samantaray and N. Mallick, “Production and characterization of poly-β-hydroxybutyrate (PHB) polymer from Aulosira fertilissima,” Journal of Applied Phycology, vol. 24, no. 4, pp. 803–814, 2012.
View at: Publisher Site | Google Scholar
K. H. Ogbonda, R. E. Aminigo, and G. O. Abu, “Influence of temperature and pH on biomass production and protein biosynthesis in a putative Spirulina sp,” Bioresource Technology, vol. 98, no. 11, pp. 2207–2211, 2007.
View at: Publisher Site | Google Scholar

Copyright

Copyright © 2014 Roberta Guimarães Martins et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

PDF Download Citation

Download other formats

Order printed copies

Views

3601

Downloads

1528

Citations